The tree shrew (Tupaia glis) has been described as a missing link relating primate to insectivore stock. The pineal gland of the tree shrew consists of a superficial pineal and a deep pineal, which are connected by a long and slender pineal stalk. A monoclonal antibody against leu-enkephalin was used in an immunohistochemical investigation of the tree shrew pineal gland. A moderate innervation of leu-enkephalin immunoreactive nerve fibers has been demonstrated in both superficial and deep pineal gland of the tree shrew. The density of the nerve fibers was slightly higher in the superficial pineal than that of the deep one. The number of immunoreactive nerve fibers were observed in the capsule of the pineal gland from where they entered the pineal parenchyma. Only a few immunoreactive fibers were found in the habenular area and the area rostral to the pineal recess, connecting the habenula and the deep pineal. Furthermore, some positive fibers were located in the pineal stalk. There was no evidence of leu-enkephalin immunoreactive intrapineal cells as seen in the other species of mammal. Therefore, the interspecies variation of opioidergic innervation among the mammals may exist. The lack of intrapineal perikarya is interpreted to indicate that the sources of leu-enkephalin nerve fibers were outside the gland. The anatomical location of the leu-enkephalin immunoreactive nerve fibers in the tree shrew pineal gland supports to both central and peripheral pinealopetal pathways in this species.